Pia Koldkjær

Analysis of gene expression from the Wolbachia genome of a filarial nematode supports both metabolic and defensive roles within the symbiosis

The α-proteobacterium Wolbachia is probably the most prevalent, vertically transmitted symbiont on Earth. In contrast with its wide distribution in arthropods, Wolbachia is restricted to one family of animal-parasitic nematodes, the Onchocercidae. This includes filarial pathogens such as Onchocerca volvulus, the cause of human onchocerciasis, or river blindness. The symbiosis between filariae and Wolbachia is obligate, although the basis of this dependency is not fully understood. Previous studies suggested that Wolbachia may provision metabolites (e.g., haem, riboflavin, and nucleotides) and/or contribute to immune defense. Importantly, Wolbachia is restricted to somatic tissues in adult male worms, whereas females also harbor bacteria in the germline. We sought to characterize the nature of the symbiosis between Wolbachia and O. ochengi, a bovine parasite representing the closest relative of O. volvulus. First, we sequenced the complete genome of Wolbachia strain wOo, which revealed an inability to synthesize riboflavin de novo. Using RNA-seq, we also generated endobacterial transcriptomes from male soma and female germline. In the soma, transcripts for membrane transport and respiration were up-regulated, while the gonad exhibited enrichment for DNA replication and translation. The most abundant Wolbachia proteins, as determined by geLC-MS, included ligands for mammalian Toll-like receptors. Enzymes involved in nucleotide synthesis were dominant among metabolism-related proteins, whereas the haem biosynthetic pathway was poorly represented. We conclude that Wolbachia may have a mitochondrion-like function in the soma, generating ATP for its host. Moreover, the abundance of immunogenic proteins in wOo suggests a role in diverting the immune system toward an ineffective antibacterial response.

The differential cardio-respiratory responses to ambient hypoxia and systemic hypoxaemia in the South American lungfish, Lepidosiren paradoxa.

Lungfishes (Dipnoi) occupy an evolutionary transition between water and air breathing and possess well-developed lungs and reduced gills. The South American species, Lepidosiren paradoxa, is an obligate air-breather and has the lowest aquatic respiration of the three extant genera. To study the relative importance, location and modality of reflexogenic sites sensitive to oxygen in the generation of cardio-respiratory responses, we measured ventilatory responses to changes in ambient oxygen and to reductions in blood oxygen content. Animals were exposed to aquatic and aerial hypoxia, both separately and in combination. While aerial hypoxia elicited brisk ventilatory responses, aquatic hypoxia had no effect, indicating a primary role for internal rather than branchial receptors. Reducing haematocrit and blood oxygen content by approximately 50% did not affect ventilation during normoxia, showing that the specific modality of the internal oxygen sensitive chemoreceptors is blood PO(2) per se and not oxygen concentration. In light of previous studies, it appears that the heart rate responses and the changes in pulmonary ventilation during oxygen shortage are similar in lungfish and tetrapods. Furthermore, the modality of the oxygen receptors controlling these responses is similar to tetrapods. Because the cardio-respiratory responses and the modality of the oxygen receptors differ from typical water-breathing teleosts, it appears that many of the changes in the mechanisms exerting reflex control over cardio-respiratory functions occurred at an early stage in vertebrate evolution.

Genomic diversity of the human intestinal parasite Entamoeba histolytica

BackgroundEntamoeba histolytica is a significant cause of disease worldwide. However, little is known about the genetic diversity of the parasite. We re-sequenced the genomes of ten laboratory cultured lines of the eukaryotic pathogen Entamoeba histolytica in order to develop a picture of genetic diversity across the genome.ResultsThe extreme nucleotide composition bias and repetitiveness of the E. histolytica genome provide a challenge for short-read mapping, yet we were able to define putative single nucleotide polymorphisms in a large portion of the genome. The results suggest a rather low level of single nucleotide diversity, although genes and gene families with putative roles in virulence are among the more polymorphic genes. We did observe large differences in coverage depth among genes, indicating differences in gene copy number between genomes. We found evidence indicating that recombination has occurred in the history of the sequenced genomes, suggesting that E. histolytica may reproduce sexually.ConclusionsE. histolytica displays a relatively low level of nucleotide diversity across its genome. However, large differences in gene family content and gene copy number are seen among the sequenced genomes. The pattern of polymorphism indicates that E. histolytica reproduces sexually, or has done so in the past, which has previously been suggested but not proven.

Seasonality of the red blood cell stress response in rainbow trout(Oncorhynchus mykiss)

SUMMARY The β-adrenergic stress response in red blood cells (RBCs) of rainbow trout shows seasonal changes in expression. We have explored the mechanisms underpinning this response by following, over a period of 27 months, changes in β-adrenergic receptor (β-AR) binding characteristics,β -adrenergically stimulated RBC Na+/H+ exchanger (βNHE) activity, together with β-AR and βNHE mRNA levels and plasma steroid hormone and lactate levels. These parameters were measured at approximately monthly intervals in a single population of fish held under semi-natural conditions. Membrane-bound, high-affinity β-ARs were present in RBCs at all sampling times, varying from 668±112 receptors cell-1 to 2654±882 receptors cell-1 (mean± s.e.m.; N=8). βNHE activity, however, was reduced by 57% and 34% in December 1999 and February 2001, respectively, compared with an otherwise sustained influx that averaged 110.4±2.3 mmol l-1 RBCs h-1 (N=119). Only one reduction coincided with a spawning period but both were preceded by transient increases in circulating testosterone. βNHE activity measured under standard conditions was not correlated with the number or affinity of β-ARs nor with water temperature, but both β-AR numbers and βNHE activity were positively related to their respective mRNA levels (P=0.005 and 0.038, respectively). Pharmaceutical intervention in the transduction cascade linking the β-AR and βNHE failed to indicate any failure of the transduction elements in RBCs displaying low βNHE activity. Similarly, we failed to demonstrate any link between seasonal cortisol fluctuations and seasonally reduced βNHE activity. However, the βNHE activity of age-separated RBC fractions showed that younger RBCs had a significantly higher βNHE response than older RBCs, consistent with the seasonal reductions in βNHE being linked to turnover of RBCs and erythropoiesis. Testosterone is known to induce erythropoiesis and we conclude that seasonal reductions in βNHE are not caused by changes in β-AR numbers but may be linked to testosterone-induced erythropoiesis.

Adrenergic receptors, Na+/H+ exchange and volume regulation in lungfish erythrocytes.

Abstract. Aestivation in African and South American lungfish (Protopterus and Lepidosiren, respectively) is associated with elevations of extracellular osmolarity. Osmotic shrinkage of Protopterus red blood cells (RBCs) caused a small but significant stimulation of the Na influx that was amiloride-sensitive, suggesting involvement of the Na+/H+ exchanger (NHE). The associated in vitro regulatory volume increase was insignificant within a time frame of 120xa0min, but the shrinkage-activated Na+ influx may be sufficient for slow regulatory volume increase during aestivation in vivo. Osmotic swelling of the RBCs induced an incomplete regulatory volume decrease that was statistically significant after 180xa0min. The RBCs of Protopterus were very large (mean cellular volume of 6939±294xa0µm3) and possessed 23066±7326 β-adrenoceptors cell–1 with a Kd value of 6.1±3.2xa0nM. The number of receptors per unit surface area of lungfish RBCs was calculated to be twice that of trout RBCs and 70% that of cod RBCs. There was, however, no adrenergic stimulation of the NHE in either Protopterus or Lepidosiren. Acidification of the extracellular medium also failed to activate the NHE.

In vivo red blood cell sickling and mechanism of recovery in whiting, Merlangius merlangus

SUMMARY Haemoglobin concentrations in vertebrate red blood cells are so high that in human sickle cell disease a single surface amino acid mutation can result in formation of large insoluble haemoglobin aggregates at low oxygen levels, causing peculiar cell deformations or `sickling. This may cause vascular occlusion and thereby severe pain, organ failure and death. Here, using light and transmission electron microscopy, we demonstrate extensive in vivo sickling of whiting red blood cells after capture stress without any apparent haemolysis and show its subsequent recovery. We show exceptionally high cooperative proton binding during the sickling process in vitro and identify the reduction of extracellular pH below resting values as the primary cause for in vivo sickling, although the response is modulated to a lesser extent also by oxygen tension. Using isotope tracer fluxes, we further show that β-adrenergic hormones, which are released under capture stress, activate a powerful endogenous Na/H exchanger in these fish red blood cells, which is known to elevate intracellular pH.β -adrenergic treatment further leads to a marked reduction of acid-induced in vitro sickling, which is impaired when Na/H exchange is inhibited by amiloride. We propose that this mechanism protects red blood cells of some fishes against the problem of haemoglobin aggregation and red blood cell sickling, except under most severe acidosis. This system offers a unique example of how, over evolutionary time, nature may have overcome what is still a deadly disease in humans.

'Two different oxygen sensors regulate oxygen sensitive K+ transport in crucian carp red blood cells'

The O2 dependence of ouabain‐independent K+ transport mechanisms has been studied by unidirectional Rb+ flux analysis in crucian carp red blood cells (RBCs). The following observations suggest that O2 activates K+–Cl− cotransport (KCC) and deactivates Na+–K+–2Cl− cotransport (NKCC) in these cells via separate O2 sensors that differ in their O2 affinity. When O2 tension (PO2) at physiological pH 7.9 was increased from 0 to 1, 4, 21 or 100 kPa, K+ (Rb+) influx was increasingly inhibited, and at 100 kPa amounted to about 30% of the value at 0 kPa. This influx was almost completely Cl− dependent at high and low PO2, as shown by substituting Cl− with nitrate or methanesulphonate. K+ (Rb+) efflux showed a similar PO2 dependence as K+ (Rb+) influx, but was about 4–5 times higher over the whole PO2 range. The combined net free energy of transmembrane ion gradients favoured net efflux of ions for both KCC and NKCC mechanisms. The KCC inhibitor dihydroindenyloxyalkanoic acid (DIOA, 0.1 mm) abolished Cl−‐dependent K+ (Rb+) influx at a PO2 of 100 kPa, but was only partially effective at low PO2 (0–1 kPa). At PO2 values between 0 and 4 kPa, K+ (Rb+) influx was further unaffected by variations in pH between 8.4 and 6.9, whereas the flux at 21 and 100 kPa was strongly reduced by pH values below 8.4. At pH 8.4, where K+ (Rb+) influx was maximal at high and low PO2, titration of K+ (Rb+) influx with the NKCC inhibitor bumetanide (1, 10 and 100 μm) revealed a highly bumetanide‐sensitive K+ (Rb+) flux pathway at low PO2, and a relative bumetanide‐insensitive pathway at high PO2. The bumetanide‐sensitive K+ (Rb+) influx pathway was activated by decreasing PO2, with a PO2 for half‐maximal activation (P50) not significantly different from the P50 for haemoglobin O2 binding. The bumetanide‐insensitive K+ (Rb+) influx pathway was activated by increasing PO2 with a P50 significantly higher than for haemoglobin O2 binding. These results are relevant for the pathologically altered O2 sensitivity of RBC ion transport in certain human haemoglobinopathies.

Minimal volume regulation after shrinkage of red blood cells from five species of reptiles.

Red blood cells (RBCs) from most vertebrates restore volume upon hypertonic shrinkage and the mechanisms underlying this regulatory volume increase (RVI) have been studied extensively in these cells. Despite the phylogenetically interesting position of reptiles, very little is known about their red cell function. The present study demonstrates that oxygenated RBCs in all major groups of reptiles exhibit no or a very reduced RVI upon approximately 25% calculated hyperosmotic shrinkage. Thus, RBCs from the snakes Crotalus durissus and Python regius, the turtle Trachemys scripta and the alligator Alligator mississippiensis showed no statistically significant RVI within 120 min after shrinkage, while the lizard Tupinambis merianae showed 22% volume recovery after 120 min. Amiloride (10(-4) M) and bumetanide (10(-5) M) had no effect on the RVI in T. merianae, indicating no involvement of the Na(+)/H(+) exchanger (NHE) or the Na(+)/K(+)/2Cl(-) co-transporter (NKCC) or insentive transporters. Deoxygenation of RBCs from A. mississippiensis and T. merianae did not significantly affect RVI upon shrinkage. Deoxygenation per se of red blood cells from T. merianae elicited a slow volume increase, but the mechanism was not characterized. It seems, therefore, that the RVI response based on NHE activation was lost among the early sauropsids that gave rise to modern reptiles and birds, while it was retained in mammals. An RVI response has then reappeared in birds, but based on activation of the NKCC. Alternatively, the absence of the RVI response may represent the most ancient condition, and could have evolved several times within vertebrates.

Magnitude of the Root effect in red blood cells and haemoglobin solutions of fishes: a tribute to August Krogh.

Aim:u2002 The ability of high carbon dioxide tensions or low pH to reduce blood oxygen binding even at high oxygen tensions, first observed by August Krogh and Isabella Leitch in 1919 and now known as the Root effect, was studied in red blood cells and haemoglobin solutions of several fish species.

Pronounced in vivo hemoglobin polymerization in red blood cells of Gulf toadfish: a general role for hemoglobin aggregation in vertebrate hemoparasite defense?

Two human hemoglobin (Hb) variants, Hb C and Hb S, are known to protect against Plasmodium falciparum malaria and have evolved repeatedly in malaria endemic areas. Both aggregate to insoluble crystals (Hb C) or polymers (Hb S) under certain physiological conditions, impair parasite growth, and may facilitate retention of infected red blood cells (RBCs) in the spleen. Given the profound effects of parasites on host evolution in general, and that RBC Hb concentration is often close to its solubility limit throughout vertebrates, similar mechanisms may operate in nonhuman vertebrates. Here we show exercise-induced, profound in vivo Hb polymerization in RBCs of the Gulf toadfish. Hb aggregation was closely associated with the extent of plasma acidosis, fully reversible, and without any signs of hemolysis or anemia. Our literature analysis suggests that aggregation prone Hbs may be relatively old, evolved multiple times in nonhuman vertebrates, show enhanced aggregation during hemoparasite infections, and can be uncovered in vivo by splenectomy. We discuss the working hypothesis that widespread Hb aggregation within several vertebrate groups may be the result of ongoing or past selection pressure against RBC parasites. Further comparative studies of these evolutionary old systems may provide valuable insights into hemoparasite susceptibility and reservoir potential of livestock and companion animals but also into human malaria and sickle cell disease.